Nucleolar dominance is an epigenetic phenomenon that occurs in plant and animal genetic hybrids and describes the transcription of ribosomal RNA (rRNA) genes inherited from only one parent due to the selective silencing of the other progenitor's rRNA genes. The silencing that occurs in nucleolar dominance happens on a scale of millions of basepairs, second in scope only to X chromosome inactivation in female mammals. Unique aspects of nucleolar dominance are that the choice of rRNA genes to silence is not random nor is it dictated by maternal or paternal imprints. The mechanisms responsible for selectively inactivating one parental set of rRNA genes in nucleolar dominance are not clear. Likewise, it is not clear how silencing decisions are made for hundreds of other genes that display monoallelic expression, for genes that are subjected to developmentally controlled silencing, or for tumor suppressor genes that become silenced in numerous forms of cancer. Therefore, understanding the molecular mechanisms responsible for nucleolar dominance has broad relevance in genetics, developmental biology and medicine. In Arabidopsis suecica, the allotetraploid hybrid of A. thaliana and A. arenosa, the A. thaliana- derived rRNA genes are silenced. Under the current grant, we identified multiple activities required for rRNA gene silencing, including a de novo DNA methyltransferase, methylcytosine binding domain proteins, histone deacetylases and histone methyltransferases. Importantly, we recently found that rRNA gene silencing is RNA and Dicer-dependent, involving components of the siRNA-directed DNA methylation pathway. Because homologous pairing of siRNAs with complementary sequences has the potential to discriminate between parental sets of rRNA genes, this discovery has the potential to reveal the choice mechanism(s) in nucleolar dominance. Moreover, siRNAs implicated in rRNA gene silencing derive from intergenic non-coding RNA (ncRNA) precursors, which are the most prevalent class of transcripts generated in the nucleus, yet the least understood. At present, we do not know whether siRNAs, their precursor transcripts, or chromatin modifications resulting from the act of intergenic spacer transcription are what mediate rRNA gene silencing, and testing these alternative hypotheses is a priority. Using a combination of genetic, cytological, bioinformatic and biochemical approaches, our short-term goals are to understand the interplay between the non-coding intergenic RNAs and the chromatin modifying activities that are involved in rRNA gene silencing. These efforts will contribute to our long-term goals of understanding the mechanisms responsible for nucleolar dominance and the mechanisms capable of silencing chromosomal loci on a megabase scale. PUBLIC HEALTH RELEVANCE: Gene silencing plays an important role in controlling the sets of genes that are repressed in specific cell types during development, in controlling the dosage of X-linked genes in mammals and in determining the parent-specific expression of imprinted genes in animals and plants. Gene silencing also plays an important role in genome defense by suppressing the activity of transposons and retroviruses. However, gene silencing has a dark side, including the silencing of tumor suppressor genes in many forms of cancer. An ability to specifically derepress tumor suppressor genes would be desirable as a cancer treatment. Likewise, the ability to derepress specific members of multigene families would be desirable as a treatment for certain genetic disorders. For instance, thalassemias that result from mutations in adult-specific globin genes might be alleviated by derepression of functional globin genes that were expressed during fetal development but were then developmentally silenced. Understanding the mechanisms responsible for gene silencing therefore has relevance to basic science as well as medicine. The unique aspects of rRNA gene silencing in nucleolar dominance will contribute to this understanding.